Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks

Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.Homologous recombination (HR) is often described as a double-edged sword: it can maintain genome stability by promoting DNA repair, while its injudicious action can disturb genome stability by causing gross chromosome rearrangement (GCR) or loss of heterozygosity (LOH). Both GCR and LOH are potential precursors of diseases such as cancer, and consequently there is need to control when and how HR is used.A key step in most HR is the loading of the Rad51 recombinase onto single-stranded DNA (ssDNA), which forms a nucleoprotein filament (nucleofilament) that catalyzes the pairing of homologous DNAs and subsequent strand invasion (32). This is a critical point at which recombination can be regulated through the removal of the Rad51 filament (60). Early removal can prevent strand invasion altogether, freeing the DNA for alternative processing. Later removal may limit unnecessary filament growth, free the 3′-OH of the invading strand to prime DNA synthesis, and ultimately enable ejection of the invading strand, which is important for the repair of double-strand breaks (DSBs) by synthesis-dependent strand annealing (SDSA). SDSA avoids the formation of Holliday junctions that can be resolved into reciprocal exchange products (crossovers), which may result in GCR or LOH if the recombination is ectopic or allelic, respectively.One enzyme that appears to be able to control Rad51 in the aforementioned manner is the yeast superfamily 1 DNA helicase Srs2 (42). In Saccharomyces cerevisiae, Srs2 is recruited to stalled replication forks by the SUMOylation of PCNA, and there it appears to block Rad51-dependent HR in favor of Rad6- and Rad18-dependent postreplication repair (1, 2, 35, 50, 53, 58). In vitro Srs2 can strip Rad51 from ssDNA via its DNA translocase activity (31, 62) and therefore probably controls HR at stalled replication forks by acting as a Rad51 disruptase. In accord with this, chromatin immunoprecipitation analysis has shown that Rad51 is enriched at or near replication forks in an srs2 mutant (50). Srs2 also plays an important role in crossover avoidance during DSB repair, where it is thought to promote SDSA by both disrupting Rad51 nucleofilaments and dissociating displacement (D) loops (20, 27).Srs2 is conserved in the fission yeast Schizosaccharomyces pombe (19, 43, 63) and has a close relative in bacteria called UvrD, which can similarly control HR by disrupting RecA nucleofilaments (61). However, an obvious homologue in mammals has not been detected. Recently, two mammalian members of the RecQ DNA helicase family, BLM and RECQL5, were shown to disrupt Rad51 nucleofilaments in vitro (11, 25), although in the case of BLM, this activity appears to be relatively weak (5, 55). Nevertheless these data have led to speculation that both BLM and RECQL5 might perform a function similar to that of Srs2 in vivo (6). Certainly mutational inactivation of either helicase results in elevated levels of HR and genome instability, with an associated increased rate of cancer (23, 25). However, BLM and RECQL5 are not the only potential Rad51 disruptases in mammals; a relative of Srs2 and UvrD called FBH1 was recently implicated in this role by genetic studies of its orthologue in S. pombe and by its ability to partially compensate for the loss of Srs2 in S. cerevisiae, which, unlike S. pombe, lacks an FBH1 orthologue (15). FBH1 is so named because of an F box near its N terminus—a feature that makes it unique among DNA helicases (28). The F box is important for its interaction with SKP1 and therefore the formation of an E3 ubiquitin ligase SCF (SKP1-Cul1-F-box protein) complex (29). The targets of this complex are currently unknown. In S. pombe, mutations within Fbh1''s F-box block interaction with Skp1 and prevent Fbh1 from localizing to the nucleus and forming damage-induced foci therein (57). Fbh1''s role in constraining Rad51 activity in S. pombe is evidenced by the increase in spontaneous Rad51 foci and accumulation of UV irradiation-induced Rad51-dependent recombination intermediates in an fbh1Δ mutant (47). Moreover, loss of both Fbh1 and Srs2 in S. pombe results in a synergistic reduction in cell viability, and like Srs2, Fbh1 is essential for viability in the absence of the S. pombe RecQ family DNA helicase Rqh1, which processes recombination intermediates (47, 48). In both cases the synthetic interaction is suppressed by deleting rad51, suggesting that Fbh1 works in parallel with Srs2 and Rqh1 to prevent the formation of toxic recombination intermediates. In yeast, Rad51-mediated recombination is dependent on Rad52 (Rad22 in S. pombe), which is believed to promote the nucleation of Rad51 onto DNA that is coated with the ssDNA binding protein replication protein A (RPA) (18, 32). Intriguingly, the genotoxin sensitivity and recombination deficiency of a rad22 mutant are suppressed in a Rad51-dependent manner by deleting fbh1 (48). This suggests that Fbh1 and Rad22 act in opposing ways to modulate the assembly of the Rad51 nucleofilament. Although current data indicate a role for Fbh1 in controlling HR, the only evidence so far that Fbh1 limits recombinant formation is in chicken DT40 cells, for which a modest increase in sister chromatid exchange has been noted when FBH1 is deleted (30).Here we present in vivo evidence suggesting that Fbh1 does indeed act as a Rad51 disruptase, which is dependent on its DNA helicase/translocase activity. We confirm predictions that this activity works in opposition to Rad22 for the loading of Rad51 onto DNA and show that Fbh1''s modulation of Rad51 activity, while not essential for limiting spontaneous direct-repeat recombination, is critical for preventing recombination at blocked replication forks. Finally, we highlight similarities and differences between Fbh1 and Srs2, based on their mutant phenotypes and relative abilities to suppress recombination when overexpressed. Overall our data affirm that Fbh1 is one of the principal modulators of Rad51 activity in fission yeast and therefore may play a similar role in vertebrates.     

真核细胞染色体DNA复制起始及复制叉稳定性维持的机制

在真核生物中,DNA复制在染色体上特定的多位点起始．当细胞处在晚M及G1期,多个复制起始蛋白依次结合到DNA复制源,组装形成复制前复合体．pre．RC在Gl-S的转折期得到激活,随后,多个直接参与DNA复制又形成的蛋白结合到DNA复制源,启动DNA的复制,形成两个双向的DNA复制又．在染色体上,移动的DNA复制又经常会碰到复制障碍（二级DNA结构、一些蛋白的结合位点、损伤的碱基等）而暂停下来,此时,需要细胞周期检验点的调控来稳定复制叉,否则,会导致复制又垮塌及基因组不稳定．本文就真核细胞染色体DNA复制起始的机制,以及复制又稳定性的维持机制进行简要综述．     

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

Many replication proteins assemble on the pre-RC-formed replication origins and constitute the pre-initiation complex (pre-IC). This complex formation facilitates the conversion of Mcm2–7 in the pre-RC to an active DNA helicase, the Cdc45–Mcm–GINS (CMG) complex. Two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), work to complete the formation of the pre-IC. Each kinase is responsible for a distinct step of the process in yeast; Cdc45 associates with origins in a DDK-dependent manner, whereas the association of GINS with origins depends on CDK. These associations with origins also require specific initiation proteins: Sld3 for Cdc45; and Dpb11, Sld2, and Sld3 for GINS. Functional homologs of these proteins exist in metazoa, although pre-IC formation cannot be separated by requirement of DDK and CDK because of experimental limitations. Once the replicative helicase is activated, the origin DNA is unwound, and bidirectional replication forks are established.The main events at the initiation step of DNA replication are the unwinding of double-stranded DNA and subsequent recruitment of DNA polymerases, to start DNA synthesis. Eukaryotic cells require an active DNA helicase to unwind the origin DNA. The core components of the replicative helicase, Mcm2–7, are loaded as a head-to-head double hexamer connected via their amino-terminal rings (Evrin et al. 2009; Remus et al. 2009; Gambus et al. 2011) onto Orc-associated origins, to form the pre-RC in late M and G₁ phases (see Bell and Kaguni 2013). However, Mcm2–7 alone does not show DNA helicase activity at replication origins. After the formation of the pre-RC, other replication factors assemble on origins, and the pre-initiation complex (pre-IC) is formed. The pre-IC is defined as a complex formed just before the initiation of DNA replication (Zou and Stillman 1998); in yeast, it contains at least seven additional factors: Cdc45, GINS, Dpb11, Sld2, Sld3, Cdc45, and DNA polymerase ε (Pol ε) (Muramatsu et al. 2010). The formation of the pre-IC is a prerequisite for the activation of the Mcm2–7 helicase; two additional factors, Cdc45 and GINS, associate with Mcm2–7 and form a tight complex, the Cdc45–Mcm–GINS (CMG) complex (Gambus et al. 2006; Moyer et al. 2006). This reaction requires components of the pre-IC and two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) (for reviews, see Labib 2010; Masai et al. 2010; Tanaka and Araki 2010). In this article, we summarize and discuss the manner via which the pre-IC is formed in yeasts and metazoa. Although there are some discrepancies, the process of formation of the pre-IC is conserved fairly well in these organisms.     

SLFN11 Blocks Stressed Replication Forks Independently of ATR

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Stabilization of Reversed Replication Forks by Telomerase Drives Telomere Catastrophe

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Substrate-Selective Repair and Restart of Replication Forks by DNA Translocases

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Recovery of Arrested Replication Forks by Homologous Recombination Is Error-Prone

Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.     

Sen1 Is Recruited to Replication Forks via Ctf4 and Mrc1 and Promotes Genome Stability

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Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks

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Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand

DNA damage bypass pathways promote the replication of damaged DNA when replication forks stall at sites of DNA damage. Template switching is a DNA damage bypass pathway in which fork-reversal helicases convert stalled replication forks into four-way DNA junctions called chicken foot intermediates, which are subsequently extended by replicative DNA polymerases. In yeast, fork-reversal is carried out by the Rad5 helicase using an unknown mechanism. To better understand the mechanism of Rad5 and its specificity for different fork DNA substrates, we used a FRET-based assay to observe fork reversal in real time. We examined the ability of Rad5 to bind and catalyze the reversal of various fork DNA substrates in the presence of short gaps in the leading or lagging strand as well as in the presence or absence of RPA and RNA primers in the lagging strand. We found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates with 5′ overhangs that can be extended by replicative DNA polymerases during the subsequent steps of template switching.     

FANCD2-Controlled Chromatin Access of the Fanconi-Associated Nuclease FAN1 Is Crucial for the Recovery of Stalled Replication Forks

Fanconi anemia (FA) is a cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand cross-links (ICLs). Within the FA pathway, an upstream core complex monoubiquitinates and recruits the FANCD2 protein to ICLs on chromatin. Ensuing DNA repair involves the Fanconi-associated nuclease 1 (FAN1), which interacts selectively with monoubiquitinated FANCD2 (FANCD2^Ub) at ICLs. Importantly, FANCD2 has additional independent functions: it binds chromatin and coordinates the restart of aphidicolin (APH)-stalled replication forks in concert with the BLM helicase, while protecting forks from nucleolytic degradation by MRE11. We identified FAN1 as a new crucial replication fork recovery factor. FAN1 joins the BLM-FANCD2 complex following APH-mediated fork stalling in a manner dependent on MRE11 and FANCD2, followed by FAN1 nuclease-mediated fork restart. Surprisingly, APH-induced activation and chromatin recruitment of FAN1 occur independently of the FA core complex or the FAN1 UBZ domain, indicating that the FANCD2^Ub isoform is dispensable for functional FANCD2-FAN1 cross talk during stalled fork recovery. In the absence of FANCD2, MRE11 exonuclease-promoted access of FAN1 to stalled forks results in severe FAN1-mediated nucleolytic degradation of nascent DNA strands. Thus, FAN1 nuclease activity at stalled replication forks requires tight regulation: too little inhibits fork restart, whereas too much causes fork degradation.     

Replication Fork Barriers and Topological Barriers: Progression of DNA Replication Relies on DNA Topology Ahead of Forks

During replication, the topology of DNA changes continuously in response to well-known activities of DNA helicases, polymerases, and topoisomerases. However, replisomes do not always progress at a constant speed and can slow-down and even stall at precise sites. The way these changes in the rate of replisome progression affect DNA topology is not yet well understood. The interplay of DNA topology and replication in several cases where progression of replication forks reacts differently to changes in DNA topology ahead is discussed here. It is proposed, there are at least two types of replication fork barriers: those that behave also as topological barriers and those that do not. Two-Dimensional (2D) agarose gel electrophoresis is the method of choice to distinguish between these two different types of replication fork barriers.     

Chaperoning HMGA2 Protein Protects Stalled Replication Forks in Stem and Cancer Cells

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Minimal Resection Takes Place during Break-Induced Replication Repair of Collapsed Replication Forks and Is Controlled by Strand Invasion

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SUMO Modification Regulates BLM and RAD51 Interaction at Damaged Replication Forks

The gene mutated in Bloom''s syndrome, BLM, is important in the repair of damaged replication forks, and it has both pro- and anti-recombinogenic roles in homologous recombination (HR). At damaged forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased γ-H2AX foci. Because the increased γ-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM''s function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess γ-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM''s pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function.     

EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.     

Csm3, Tof1, and Mrc1 Form a Heterotrimeric Mediator Complex That Associates with DNA Replication Forks

Mrc1 (mediator of replication checkpoint), Tof1 (topoisomerase I interacting factor), and Csm3 (chromosome segregation in meiosis) are checkpoint-mediator proteins that function during DNA replication and activate the effector kinase Rad53. We reported previously that Mrc1 and Tof1 are constituents of the replication machinery and that both proteins are required for the proper arrest and stabilization of replication forks in the presence of hydroxyurea. In our current study, we show that Csm3 is a component of moving replication forks and that both Tof1 and Csm3 are specifically required for the association of Mrc1 with these structures. In contrast, the deletion of mrc1 did not affect the association of Tof1 and Csm3 with the replication fork complex. In agreement with previous observations in yeast cells, the results of a baculovirus coexpression system showed that these three proteins interact directly with each other to form a mediator complex in the absence of replication forks.     

Multiple Roles of Replication Forks in S Phase Checkpoints: Sensors,Effectors and Targets

There is mounting evidence that replication defects are the major source of spontaneous genomic instability in the cell and that S phase checkpoints are the principle defense against such instability. In Saccharomyces cerevisiae, S phase checkpoints can be provoked by either depletion of dNTPs or DNA damage. In both cases the checkpoint kinases Mec1 and Rad53 act to suppress late origin firing, stabilize slowed or stalled replication forks and prevent S phase progression until conditions are appropriate for the resumption of DNA replication. The present review highlights recent work emphasizing the central importance of replication forks, not just as targets, but also as sensors and primary effectors of checkpoint responses, and identifies the roles played by specific fork-associated factors in these processes.